A systematic survey of the molecular genetic changes in primary pancreatic carcinoma has never been done. There is now the ability to do such studies, taking advantage of the large number of resections for pancreatic carcinoma done at The John Hopkins Hospital, a frozen tissue bank containing 82 fresh frozen pancreatic adenocarcinomas and normal tissues from resections, the development of cryostat dissection, cell culture, and xenograft methods to enrich for neoplastic cells, and a multifaceted approach to analyzing both the genome as well as selected genes for genetic alterations. This project embarks upon a comprehensive study of the genetics of pancreatic cancer and the clinical usefulness of this knowledge. Specifically, the project will 1) determine the ras, p53, and APC mutations by sequencing DNA of cryostat-enriched primary pancreatic exocrine adenocarcinomas, xenografts, and cell lines, 2) determine the sites of candidate tumor suppressor genes by dinucleotide repeat PCR to evaluate for allelic loss at over 300 well-spaced loci per tumor, 3) screen for genetic rearrangements and deletions by a whole genome subtraction approach, the representational difference analysis (RDA), and 4) screen for gene amplifications by Southern blot technique using probes for over 35 known oncogenes and tumor suppressor genes and by comparative genomic hybridization (CGH) using fluorescent in-situ hybridization. Sensitivity and confirmation will be enhanced by comparing results of multiple independent xenograft tumors with the respective primary carcinoma tissue. Results will be correlated with clinical and pathological databases. Extensive interdepartmental and interinstitutional collaborations will be fostered. Aspects of these studies have direct application to the companion project 1.